Since the discovery of the DNA molecules by Watson and Crick in the 50's, huge scientific progress has been made within the field of biochemistry and biology. Recombinant DNA techniques and techniques for genetic manipulation are continuously developped based on the new discoveries and new medical applications thereof are proposed every day. In general, such methods are based on the principle that some kind of genetic information containing material, such as a gene, is transferred from its original and native environment to a new environment, where some desired properties thereof may be utilized in a new context. Usually, some kind of carrier or vector is used for such a transfer procedure, of which one example is virus. Viruses are due to the native properties thereof capable of infecting cells and thereby transferring its own contents of nucleic acids thereto. Successful genetic manipulation requires appropriate reagents, such as vectors, as well as practical and effective procedures for the performance thereof. As one example of a medicinal application, which is of utmost interest at the moment, may gene therapy be mentioned. In gene therapy, vectors, such as virus particles, are used to introduce nucleic acid sequences in receptor cells, where said sequences may compensate for damages or mutations, or even correct mutations, in the native genes of said cells.
During the elaboration of better and more efficient recombinant techniques, it has appeared that the cells which are desirable as receptor or target cells often are quite difficult to infect. The simplest and most straight forward approach to enhance the number of infected cells is to increase the amount of virus used during the infection procedure, whereby the number of completed infection occurences in receptor cells hopefully increase as well. The result of this approach is, however, an increased demand for cells producing virus in high concentrations. Problems arise, however, when cells are to produce large amounts of a virus. In many cases, the virus is toxic to the producer cells when present in such massive amounts, and, accordingly, the cells are killed when the virus production is increased. In cases where large foreign nucleic acid sequences have been introduced in the virus, the virus particles may not be assembled in a correct way during a large scale production.
Different ways of transfecting cells with vectors, such as viruses, have been proposed. One procedure is disclosed, where a suspension of target cells is held in a container, whereafter a virus containing cell medium is added thereto. However, since a virus is a colloidal particle, it will move randomly with Brownian motions in a liquid. Accordingly, the transfection takes place randomly in such a procedure, depending on the amount of particles that eventually hit the cells. Only a small fraction of the virus particles will ever reach contact with target cells and, in addition, the contact area and binding achieved is usually too loose to permit a sufficient transfection. The virus containing cell suspension may be stirred or agitated in order to increase the efficacy of such a process. Still, the efficiency of the process is low and not optimal for an industrial method.
In order to increase the contact area between target cells and virus, and thus increase the number of infection occurences, target cells have been immobilized on membranes. Then, a virus containing supernatant is passed through the membrane. Indeed, a better contact between virus particles and cells than in the case described above may be obtained. However, such a membrane technique will exhibit other essential drawbacks. One problem when using a membrane in connection with cells is that it will sooner or later be obstructed by clogging cells. Thus, the life-time of such a membrane and also the possible choices of flow conditions are limited. Another problem results from the fact, that the virus containing supernatant must be forced against the target cells on the solid membrane, whereupon the virus binding receptors on the target cells are often damaged. Consequently, the possible binding of virus theron will be reduced. An additional drawback with the membrane techniques originates from the fact that only a limited number of cells may be immobilized on each membrane, which number is restricted by the area of the membrane. In fact, preliminary calculations reveal that to attain a sufficiently large amount of cells for clinical experiments the diameter of such a membrane need to be about 34 m. In addition, the amount of supernatant needed to wet such an area is enormous. Naturally, such a large membrane is hardly applicable in practice.
Other cellular filters have been proposed for similar applications. In Biotechnol. Prog. 1990, 6, p. 104-113, by Krishna Mohan et al, a cellular filter is disclosed for the removal of trace viral contaminants in blood. However, the aim of these authors is to purify blood or similar fluids, and therefore, the properties of the resulting fluid are stressed, while no particular account is taken to protect or enhance the cells that absorb the virus. In this publication, one strategy is disclosed, where cells are grown on microcarriers and stabilized by a cross-linking agent. Consequently, a cellular filter is accomplished. Said filter will exhibit properties similar to the above described membranes with immobilized cells thereon, i.e. it will constitute an almost stable element against which cells absorbing virus will be forced. Another strategy disclosed in this article involves the use of clusters or aggregates of cells around microcarriers. The microspheres used to cluster the cells exhibits a magnetic nature, which is then utilized combined with other magnetic equipment around the reaction vessel. Obviously, such a magnetic agitation will be efficient for the application disclosed in the article, i.e. the recovery of a virus free solution. However, in the instance when the infected cells are the products to be used for later applications, it will result in too large a cell desintagration and, in addition, the magnetic field may be harmful as regards internal properties of the absorbing cells.